Ebola virus is a member of the Filoviridae family of viruses. There are 5 species of Ebola virus and some of these are associated with fatality rates of up to 90%. As has been highlighted during the recent outbreak of Ebola Zaire (EBOV) in Western Africa, filoviruses have tremendous potential for adverse health effects both directly (through pathogenesis) and indirectly (forcing the shutdown of existing health clinics and treatment programs, sparking panic worldwide). The glycoprotein (GP) of EBOV is the protein involved in virus entry and has extensively investigated. GP has been suggested to be important for determining virulence of the virus and has been associated with immune evasion through the production of sGP. GP has also been used as part of efforts to provide protection against EBOV. Non-pathogenic viruses (such as vesicular stomatitis virus, the rabies vaccine, chimp adenovirus type 3, etc) that express EBOV GP have been shown to provide protection against EBOV in animal models of disease. This places GP as a molecule that plays a critical role in both virus replication and vaccine efficacy. Much has been learned about the function of GP, but much remains to be discovered. In this proposal we will investigate how mutations in GP impact virus (and vaccine) replication efficiency. To do this we will use a forward genetic approach using a recombinant vesicular stomatitis virus that encodes the EBOV GP from one of the initial Makona virus isolates as its only glycoprotein. We will then track the appearance of spontaneous mutants within the population of this virus to identify mutations within GP that increase virus replication efficiency. These mutations will be mapped on to known mutations that occur within the EBOV GP in ?hot?? virus both during outbreaks and during tissue culture adaptation to identify identical mutations. These mutations will be analyzed mechanistically to understand how mutations increase entry efficacy. These studies will allow us to us a selective-advantage based mutation approach to identify residues critical for EBOV GP function. These findings will provide mechanistic insight into the ?meaning? of mutations already known to occur in EBOV stocks used in challenge studies. They will also provide important information with regards to approaches for increasing production of potential vaccines based on EBOV GP and will provide insight into the entry process of this complicated viral protein.